Studies on DNA replication of plasmid Co1E1 have been continued. Transcripts (RNA II) that start 555 nucleotides upstream of the replication origin by RNA polymerase form hybrids with the template DNA. The hybridized transcript is cleaved by ribonuclease H at the origin and used as the primer for leading strand synthesis of DNA polymerase I. Primer formation is regulated by a plasmid-specified small RNA (RNA I), which is transcribed from the DNA coding 51 end region of RNA II, but in the direction opposite to that of RNA II synthesis. This antisense RNA binds to RNA II and prevents RNA II to form the secondary structure necessary for primer formation. The binding begins with interaction between loops of RNAs. Isolated complementary single stem-loops of these RNAs also form complex, in which all the bases in the loops form base-pairing. The stability is determined by both the base-pairing between loops and the basestacking with neighboring bases. Primer formation is also regulated by a 63-amino acid protein specified by the plasmid. The protein called Rom binds to a very unstable initial complex made between RNA I and RNA II and thus enhances the inhibitory action of RNA I A single dimer molecule of Rom binds to the complex formes by complementary single stem-loops by recognizing the structure rather than its exact nucleotide sequence. The binding of Rom stabilizes the complex by decreasing the rate of dissociation of the complex without affecting the rate of association of the RNA components. Inhibition of primer formation by RNA I in the presence or absence of the protein determines the copy number of a plasmid in a cell and the incompatibility between related plasmids.